Abstract 2200 Combinatorial phosphorylation patterns of the potassium ion channel hERG affect expression levels in the HEK-293 model system

The alpha subunit proteins of cardiac voltage-gated K+ ion channels, hERG and KvLQT1, contribute to the repolarization phase of action potentials in ventricular cardiomyocytes.The channels they form carry the IKr and IKs currents respectively.Disruption of these currents can manifest as prolonged action potential durations, clinically described as Long QT syndrome (LQTS).In LQTS patients, stimulation of the sympathetic nervous system through the b-adrenergic signaling pathway may lead to arrhythmia or sudden cardiac death.Our lab investigates the downstream effects of this pathway including whether and how hERG and KvLQT1 are regulated via protein-kinase A (PKA) mediated phosphorylation.The effects of combinatorial phosphorylation patterns on the biogenesis, trafficking, expression of hERG and KvLQT1 individually as well as its role in their protein-protein interactions remain unknown.We investigated how combinatorial phosphorylation in the four putative PKA sites affects the maturation and abundance of hERG relative to wild type control using transient transfection of hERG-mEGFP phosphomutants in HEK 293 cells followed by immunoblotting.Imaging of transient transfections indicates some phosphomutants show low levels of expression while phosphonull transfections are similar to wild type.Immunoblots results also show over-expression of some phosphomutants relative to wild type.This suggests that PKA mediated phosphorylation is a mechanism to regulate expression levels of hERG.